Spinning force apparatus

ABSTRACT

A spinning force system and methods of operation are provided for measuring a characteristic of a sample. The system includes a detection module having a light source for illuminating the sample and an objective being aligned to the light source to define a light path for producing an image of the sample. A rotor is mechanically coupled to the detection module and configured to rotate the light path for applying a force to the sample. The force may include a centrifugal force and other forms of force (such as a viscous drag force) resulted from the rotation. In some examples, the force is applied in a direction that is not parallel to a surface of the sample.

BACKGROUND

This invention relates to measurement of forces relating to molecules.

The ability to quantify interactions between biomolecules is of greatinterest for scientific and medical research, as well as for drugdevelopment. Examples of measurable characteristics of a biomolecularinteraction include the affinity (e.g., how strongly the moleculesbind/interact) and the kinetics (e.g., rates at which the associationand dissociation of molecules occur) of the interaction. Traditionally,such characteristics are measured in solution, using methods such ascalorimetry, stop-flow imaging, or surface plasmon resonance. These bulkmeasurements are limited in many ways, including 1) they report onlyaverage behavior and thus may lose important details associated withmetastable states and rare events, and 2) they measure chemistry in theabsence of externally applied mechanical stress, which can bedramatically different from crowded and dynamic environments in livingsystems.

Recent development in single molecule measurement methods offers adifferent approach in quantifying molecular interactions by examiningthe behavior of individual molecules rather than measuring theproperties of bulk solutions. This approach enables the observation ofrare or fleeting events that can be obscured by ensemble averaging. Theresulting detailed information of molecular transitions helpsresearchers to identify metastable states and to study the transitionsrates and the chemical pathways between such states. Furthermore,heterogeneities between molecules in a population and within thebehavior of a single molecule can both be quantified.

Currently, force probes that apply single molecule measurement methodsinclude atomic force microscopes (AFM), optical traps, magnetictweezers, biomembrane force probes, and flow chambers. Despite manyadvantages, these devices still have several limitations. For example,due to technical complexities, some systems require a large investmentof money and time (e.g. optical trap systems typically cost $150 k ormore). Additionally, molecular interactions are studied one molecule ata time in most cases. Statistical characterization of these interactionsis therefore slow and painstaking, requiring hundreds or thousands ofmeasurements which are typically performed in a serial manner.

SUMMARY

One general aspect of the invention provides a method for rotating anobject attached to a surface of a substrate. The substrate is coupled toa rotary member. The rotary member is rotated to apply a force to theobject in a direction that is not in parallel to the surface of thesubstrate. A light source illuminates the surface of the substrate. Arelative motion of the object to the surface of the substrate caused bythe force is detected using a detector.

Embodiments of this method may include one or more of the followingfeatures.

The force applied to the object may have a force component in adirection perpendicular to the surface of the substrate. The force mayinclude a centrifugal force. When the object is in contact with a liquidmedium, the force may also include a viscous drag force.

A magnitude of the force applied to the object may be controlled, forexample, by changing an angular velocity of the rotary member or bychanging a distance of the object to a center of the rotation. Thedirection of the force applied to the object may also be controlled, forexample, by changing an orientation of the substrate relative to therotary arm.

The light source and/or the detector may be rotated at an angularvelocity substantially the same as that of the rotary member. The rotarymember may be rotated about an axis substantially parallel to thesurface of the substrate.

The relative motion of the object to the surface of the subject may besufficient to detach the object from the surface of the substrate.

A magnitude of a force interaction between the object and the surface ofthe substrate may be determined based on the magnitude of the forceapplied to the object by the rotation.

The object may include a molecule. The relative motion of the object mayinclude a motion indicative of a conformational change of the molecule.The conformational change may include unfolding, refolding, stretching,and/or relaxing of the molecule. The relative motion of the object mayalso include a motion indicative of a rupture or a formation of a bondat least partially associated with the molecule.

An image of the object may be generated with the detector, and a changein the image may be determined. The image may include a transmittedlight image, a reflected light image, and/or a fluorescence image.

The detector may include a light detector, which may be a charge-coupleddevice or a CMOS detector.

The object may include a biological subject such as a molecule, a cellorganelle, a cell, or a tissue. The object may further include a carrierthat forms a chemical linkage with the biological subject. The carriermay be a bead.

The object may include a first molecule, and the surface may include asecond molecule for forming an interaction with the first molecule. Thefirst molecule may be a receptor and the second molecule may be a ligandof the receptor.

The object may include a group of individual targets attached to thesurface of the substrate. The force applied to the object may include acorresponding group of force components each applied to a respective oneof the individual targets.

Another general aspect of the invention provides a method including:attaching a particle to a surface of a substrate through a molecularinteraction associated with a first molecule and second molecule;rotating the substrate to apply a force to the particle in a directionthat is not in parallel to the surface of the substrate; detecting animage of the particle with a detector, the image containing informationrepresenting a characteristic of the molecular interaction; anddetermining the characteristic of the molecular interaction based on thedetected image.

Embodiments of this method may include one or more of the followingfeatures.

The method may further include determining a relative motion of theparticle to the surface of the substrate. The relative motion mayinclude detachment of the particle from the surface of the substrate, amotion indicative of a rupture or a formation of a chemical bondassociated with the molecular interaction, or a motion indicative of aconformational change of at least one of the first and the secondmolecules. The conformational change may include unfolding, refolding,stretching and/or relaxing of at least one of the first and secondmolecules.

Determining the characteristic of the molecular interaction may includemeasuring a strength characteristic of the molecular interaction and/ormeasuring a kinetic characteristic of the molecular interaction. Thekinetic characteristic of the molecular interaction includes anassociation rate K_(on) or a dissociation rate K_(off) of the molecularinteraction.

A first chemical linkage may be formed between the particle and thefirst molecule. A second chemical linkage may be formed between thesecond molecule and the surface of the substrate.

The method may further include controlling a rotation of the substratesuch as controlling an angular velocity of the rotation of thesubstrate.

The first molecule may be a receptor and the second molecule may be aligand of the receptor. Alternatively, the first molecule may include afirst molecular complex, and the second molecule may include a secondmolecular complex that interacts with the first molecular complex. Thefirst molecular complex may include one or more receptor-ligand pairs.

The particle may include a bead. The bead may have a diameter in therange between 1 nm and 1 mm, and a density in the range between 0.01g/cm³ and 20 g/cm³.

Another general aspect of the invention provides an apparatus formeasuring a characteristic of a sample. The apparatus includes adetection module having a light source for illuminating the sample andan objective being aligned to the light source to define a light pathfor producing an image of the sample. A rotor is mechanically coupled tothe detection module and configured to rotate the light path forapplying a force to the sample.

Embodiments of this apparatus may include one or more of the followingfeatures.

The detection module may further include a sensor for detecting theimage of the sample and for generating electrical signals representativeof the image. The sensor may include a light detector. The lightdetector may include a charge-coupled device.

The sensor may be rotated by the rotor at an angular velocity the sameas that of the light path. Alternatively, the sensor may be stationaryto a ground, in which case a rotating mirror may be provided fordirecting a light signal of the image generated by the objective to thesensor.

A positioner may be coupled to at least one of the objective and thelight source. The positioner may be configured to adjust a relativeposition of the objective to the light source.

A sample housing may be provided for mounting the sample to thedetection module. A positioner may be coupled to the sample housing. Thepositioner may be configured to adjust a configuration of the samplehousing to change an orientation of the sample, or to adjust a relativeposition of the sample housing to the objective at least in a directionof the light path. The positioner may be further configured to adjustthe relative position of the sample housing to the objective in each ofthree orthogonal directions.

A data transmission module may be coupled to the sensor. The datatransmission module may be configured to transmit the electrical signalsrepresentative of the image to be processed. The data transmissionmodule may include a first media converter for converting the electricalsignals to optical signals, a second media converter for converting theoptical signals to subsequent electrical signals to be processed, and anoptical fiber for transmitting the optical signals from the first mediaconverter to the second media converter.

A rotor controller may be coupled to the rotor. The rotor controller maybe configured to provide a control signal for controlling the rotationof the light path, or alternative, for controlling an angular velocityof the rotation of the light path.

A detection controller may be coupled to the detection module forproviding a control signal to change an optical characteristic of thedetection module. The optical characteristic may include an illuminationintensity of the light source, a light frequency of the illumination ofthe light source, and/or an image acquisition characteristic of thesensor.

Among other advantages and features, the spinning force system andmethods described herein can provide one or more of the followingadvantages.

The system and methods of operation can provide massively-parallelhigh-throughput single-molecule force measurements at a low cost. Morespecifically, rotation-induced forces (e.g., centrifugal forces andviscous drag forces) can be used to manipulate single molecules (e.g.,proteins or DNAs) or molecular complexes (e.g., receptor-ligand proteinpairs), enabling forces to be applied simultaneously to many subjects.Each subject can be observed directly and independently for truesingle-molecule detection.

The system and methods of operation can provide accurate force controlin a wide range of directions and magnitudes. Through force control, thesystem and methods of operation can be used to quantify force dependentinteractions, including measuring the force dependence of kineticparameters (e.g., K_(on) and K_(off)) and molecular subtleties whichwould be invisible from population averaging. Using this system, themechanical properties of biomolecular complexes (e.g., compliances ofDNAs and proteins) and cellular targets (e.g., elasticity ofstress-bearing cells) can be studied, yielding valuable information intoboth the structure and the function of those subjects.

The system and methods of operation can be conveniently integrated withvarious types of force probes to generate forces in multiple dimensionswith high flexibility. For example, the system can be used inconjunction with optical traps, magnetic tweezers and/or microfluidicdevices to generate a combination of forces (such as gradient andscattering forces, magnetic forces, hydrodynamic forces, and centrifugalforces). Each force can be applied to a sample in a different direction,with a different magnitude, and/or at a different test stage.

The system and methods of operation can also be conveniently integratedwith various imaging techniques to provide real-time observation withhigh temporal and spatial resolution. For example, using interferencetechniques and diffraction analysis, the position of individualparticles in a sample can be ascertained with sub-nanometer accuracy.Also, fluorescent imaging enables visualization of subtle moleculartransitions during experiment. Moreover, using video tracking byhigh-speed CCD cameras, molecular events can be detected on the scale ofmicroseconds.

Other features and advantages of the invention are apparent from thefollowing description, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of one embodiment of a spinningforce system.

FIG. 2A is a schematic representation of a sample coupled to thespinning force system of FIG. 1.

FIG. 2B shows a centrifugal force applied to the sample of FIG. 2A.

FIGS. 3A and 3B are transmitted light images of a sample generated attwo different focal depths, respectively.

FIGS. 4A-4C show the interference patterns of a sample imaged by areflection interference contrast microscope at three different heights,respectively.

FIGS. 5A-5D are schematic representations of a procedure for preparing asample to be measured by the spinning force system of FIG. 1.

FIGS. 6A-6D are schematic representations of another procedure forpreparing a sample to be measured by the spinning force system of FIG.1.

FIGS. 7A-7D are schematic representations of a further procedure forpreparing a sample to be measured by the spinning force system of FIG.1.

FIG. 8 shows an alternative embodiment (schematic in part) of thespinning force system of FIG. 1.

FIG. 9 shows an additional alternative embodiment of the spinning forcesystem of FIG. 1.

FIG. 10 shows a further alternative embodiment of the spinning forcesystem of FIG. 1.

DETAILED DESCRIPTION 1 System Overview

Referring to FIG. 1, a spinning force system 100 includes a rotary arm120 mechanically coupled to a rotary stage 110 configured to rotateabout a central axis 102 at an adjustable angular velocity ω. Rotarystage 110 is housed and supported on a stationary base 104 immobilizedon a platform (not shown) such as a vibration-free optical table.

Rotary arm 120 extends radially outward from central axis 102 to supporta set of optical, mechanical, and electrical components for detectingcharacteristics (e.g., motion, optical, and geometric characteristics)of a sample 140 to be measured by system 100. These components include,for example, a light source 130, an objective 150, a light detector 160,and a media converter 170. In operation, these components are moved byrotary arm 120 to rotate about central axis 102 at the same angularvelocity ω. Rotary arm 120 may also carry one or more positioningelements (e.g., adjustment screws and electromechanical stages such aspiezoelectric positioners) for adjusting the position of each componentcoupled to arm 120. Examples of positioning elements will be describedin greater detail below.

In this example, light source 130 is mounted at a distal end of rotaryarm 120 for emitting a light beam 132 to illuminate a selected region ofsample 140. Examples of light source 130 suitable for use in system 100include various types of lamps (such as LED bulbs and xenon arc lamps)and lasers (such as single- and multiple-wavelength lasers). Lightsource 130 may also include a set of optical components such as lenses,mirrors, and filters (not shown) for controlling the characteristics ofits outgoing beam 132. For example, a condenser with diaphragms may beused for tuning the emission intensity of beam 132, and a color filtermay be used for transmitting light at only selected wavelengths.

Sample 140 is mounted onto rotary arm 120 with a sample holder (notshown) fastened to the arm. Depending on the particular implementation,sample 140 may include a sample chamber (not shown) in which experimentsubjects (such as cells, biomolecules, and DNA strands) are sealinglycontained. The sample chamber may consist of two parallel cover glassesseparated by a 1 mm o-ring, forming an enclosed volume that can befilled with buffer and beads. In some implementations, sample 140 isoriented such that the surfaces of the cover glasses are aligned inparallel to central axis 102. When rotary arm 120 rotates, the contentsof sample 140 experience a centrifugal force normal to the coverglasses. In other implementations, sample 140 is oriented at a selected(and possibly adjustable) angle with respect to central axis 102,enabling the centrifugal force to be applied in any given direction.

Light beam 142 exiting sample 140 is received by objective 150 toproduce a real image of the illuminated region of sample 140. Theoptical characteristics of objective 150 (e.g., magnification andnumerical aperture) are selected depending on the particularimplementation. For example, a 20× air-immersion objective may be usedfor applications that require a wide field of view, whereas a 100×oil-immersion objective may be preferred for applications that require ahigh spatial resolution.

Preferably, the relative position of objective 150 with respect tosample 140 is adjustable in three dimensions (x-, y- and z-directionsshown in the figure), allowing images of different regions of the sampleto be collected at various focal depths. In this example, objective 150is staged on a piezoelectric positioner 190, which can be translatedalong each of the x-, y-, and z-directions by an external control signal(e.g., provided by a computer). In other examples, sample 140 (insteadof objective 150) may be staged on positioner 190 for linear translationin those three dimensions.

Images formed by objective 150 are received by detector 160 andsubsequently converted into electronic signals 162. One example of adetector suitable for use is a charge-coupled device (CCD), such as a12-bit 512 pixels×512 pixels CCD camera. Another example of a suitabledetector is a CMOS detector. Preferably, detector 160 is capable ofacquiring successive images at a speed sufficiently fast to enable videotracking of sample 140 at a high temporal resolution (e.g., 1 kHz). Insome examples, light from objective 150 is first transformed through anintermediate optical system (not shown) before reaching detector 160.The intermediate optical system may include one or more elements such aslenses, filters, polarizers, and pinholes.

Electronic signals 162 from detector 160 are delivered, for example,using electrical, optical, or wireless transmission means, to be passedonto a computer 180. In this example, signals 162 are transmittedsequentially through an electronic-to-optical media converter 170, anoptical fiber 174, and an optical-to-electronic media converter 172.Both media converter 172 and computer 180 are positioned on a stationaryplatform (not shown). Optionally, optical fiber 174 is coupled to rotarystage 110 through a fiber optic rotary joint (not shown), which can befurther integrated into an electrical slipring 106 of rotary stage 110.Using proper interfacing software, computer 180 decodes electronicsignals 176 from media converter 172 to reproduce images of sample 140on a screen.

Computer 180 is used for viewing and processing images of sample 140. Inaddition, computer 180 is also configured to provide various controlsignals to control individual components of spinning force system 100.For example, computer 180 may be coupled to an electric motor (notshown) for controlling a rotational drive force to change the angularspeed a) of rotary stage 110. Computer 180 may also be coupled to apositioning device (not shown) for adjusting a distance between lightsource 130 and sample 140, or coupled to positioner 190 for translatingobjective 150 in each of x-, y-, and z-directions to select detectionregions and to control focal depth. Computer 180 may also be configuredto control the optical characteristics of light source 130 (e.g., thebrightness and the frequency range of output beam 132) as well as theimage acquisition variables of detector 160 (e.g., readout rate,integration time, and electronic gain). Additionally, computer 180 mayprovide control signals based on previously acquired data, enablingreal-time feedback control.

2 Operation

2.1 Force Application

Referring to FIGS. 2A and 2B, when rotary arm 120 is in operation,sample 140 rotates at an angular velocity ω and at a distance R from thecenter of axis 102. For illustrative purposes, in this example, sample140 includes a cover slip 140′ having an inner surface 140 a and anouter surface 140 b with respect to axis 102. Both surfaces are alignedin parallel with axis 102. A particle 240 (e.g., a bead) adheres toouter surface 140 b through a chemical bond 243 formed between moleculeA 242 and molecule B 244. In this example, molecule A is a receptorchemically linked to outer surface 140 b, and molecule B is a ligandchemically linked to particle 240. (The techniques and methods forforming such linkages will be described in greater detail below).

When particle 240 undergoes circular motion, a centripetal force F isexerted on the particle, as defined by the following equation:

$\begin{matrix}{F = \frac{{mv}^{2}}{R}} & (1)\end{matrix}$where F is the net centripetal force, m and ν are the mass and thelinear velocity of the particle, respectively, and R is the distance ofthe particle from rotation axis 102. In a rotating reference frame inwhich orbiting particle 240 appears stationary, particle 240 experiencesan inertial centrifugal force equal to F in a direction perpendicular toouter surface 140 b and away from central axis 102 (shown by arrow 250).In some examples where particle 240 is a spherical bead in solution withradius r and relative density ρ, rewriting equation (1) in terms ofangular velocity ω yields:

$\begin{matrix}{F = \frac{4\;\pi\;\rho\; r^{3}R\;\omega^{2}}{3}} & (2)\end{matrix}$

When sample 140 rotates about axis 102 at a very low speed, centrifugalforce F is countered by the interaction force of chemical bond 243,allowing particle 240 to continue to adhere to surface 140 b. As therotational speed ω rises, the increasing magnitude of centrifugal forceF causes bead 240 to move with respect to surface 140 b. Thecharacteristics of the relative motion (e.g., the root-mean-squaredisplacement or the direction of the motion) can be monitored andanalyzed to quantify certain chemical and/or mechanical properties ofbond 243 (e.g., properties associated with its transitional states andconformational changes). The increasing F may also cause the rupture ofchemical bond 243, at which point, particle 240 is released from surface140 b. The magnitude of centrifugal force F at the particle releaseindicates the rupture force of chemical bond 243.

During operation, the magnitude of centrifugal force F can becontrolled, for example, by adjusting the angular velocity ω of rotaryarm 120. For instance, sample 140 can be subjected to multiple cycles offorce application in which the centrifugal force on particle 240 isincreased and/or decreased through step changes in angular velocity ω.

In addition to changing the angular velocity ω, it is also possible tochange the radius of rotation R either statically or dynamically bychanging the sample position relative to the axis of rotation. Forexample, in system 100, sample 140 may be mounted to an adjustablerotary arm with extendible length, or staged on a positioner that can betranslated in a radial direction with respect to central axis 102.

In cases where particle 240 is a spherical bead, the centrifugal force Fcan also be varied by changing one or more of the particlecharacteristics ρ and r shown in equation (2). For example, microspheresare commercially available in a wide range of materials and sizes (seeTable 1 below). By conjugating subjects of study (e.g., molecules orcells) to selected microspheres, the centrifugal force applied to themicrospheres (and translated to the subject) can be varied based on beadproperties. In addition, the degree of monodispersity of beads cancontrol the range of forces applied for a given spin. For instance, ahighly monodisperse sample (e.g., using beads of substantially the samesize and properties) may cause all beads to experience the same force,while a polydisperse sample (e.g., using beads of various sizes and/orproperties) would have a wide range of forces being applied. Moreover, ρof particle 240 can also be altered by changing the density of thebuffer solution. Furthermore, the geometry of the sample chamber can bevaried to control the effects of fluid flow, which can add hydrodynamicforces to immobilized particles in the chamber.

TABLE 1 The materials and sizes of common beads Specific Density SizeRange Bead Material (g/cm³) (μm) Borosilicate 1.5   1-100+ Polystyrene0.05  0.05-100+ Silica 1.2 0.01-100  Gold 18.3 0.002-0.25  Melamine 0.510.5-10  Iron Oxide 4.24  1-10

With proper parameter selection, the force applied to particle 240 canspan 9 orders of magnitude, ranging from microNewtons (e.g., r=10 μm,ρ=1.5 g/cm³, R=500 mm, ω=100 Hz) to femtoNewtons (e.g., r=1 μm, ρ=0.05g/cm³, R=250 mm, ω=2 Hz).

The direction of the centrifugal force F can also be controlled. In someexamples, sample 140 may be configured in an orientation perpendicularto rotational axis 102, resulting in a centrifugal force F along surface140 b. In other examples, sample may be configured to form a selectedangle with respect to rotational axis 102 so that centrifugal force Fmay be applied in any given direction. For instance, a compressive(rather than tensile) force can be applied to particle 240 if theparticle is positioned on inner surface 140 a (rather than outer surface140 b) of the cover glass. For particular implementations, it may bedesirable to place sample in a parallel position with respect torotation axis 102 because pulling particle 240 away from surface 140 breduces the likelihood of the particle forming new interactions withunoccupied binding sites of molecule A on the surface 140 b.

In addition to centrifugal force F, other types of forces can also beapplied to particle 240 through spinning. For example, if particle 240is contained in a chamber filled with a liquid medium, the rotation ofsample 140 can generate regional flows that exert a viscous drag force Dto particle 240. The direction of the drag force depends on factors suchas the geometry of the chamber and the orientation of the sample. Themagnitude of the drag force depends on factors such as the viscosity andthe temperature of the liquid medium, the size of the particle, and therotational velocity and acceleration of the sample.

2.2 Observing Motion Characteristics

In spinning force system 100, motion of particle 240 (e.g., displacementcaused by molecular folding, unfolding or rupture of bond 243) can beobserved by video tracking methods (e.g., by taking successive images ofthe particle at a high temporal resolution). Because light source 130,sample 140 and objective 150 rotate together at the same angularvelocity ω, these three components appear stationary to each other in arotating reference frame. Therefore, images of particle 240 can beformed using traditional imaging techniques, including transmitted- orreflected-light techniques and fluorescence techniques.

Referring to FIGS. 3A and 3B, images 310 and 320 are transmitted-lightimages of the same region of sample 140 produced at two different focaldepths, respectively. In this example, sample 140 contains multiplebeads (appearing, for example, as circular objects 312, 314 and 316 inimage 310) near surface 140 b. The location of each bead in the imagesrepresents the lateral position of the bead (e.g., along x- and y-axesof FIGS. 1, 3A and 3B). The image pattern of the bead (e.g., the size,the sharpness and the ring pattern of circular objects 312, 314 and 316)indicate the focal depth, namely, the distance of the bead fromobjective 150 (e.g., along z-axis of FIG. 1). This focal depth can beused to represent the relative position of the bead above surface 140 b.

By analyzing the lateral position and the image pattern of individualbeads, the movement of the beads with respect to time can becharacterized in a three-dimensional space. Such characterizationenables the quantification of the affinity and the kinetics of chemicalbond 243 that links the bead to surface 140 b. For example, beforespinning, the position of objective 150 can be adjusted such thatparticle 240 situates in the objective's focal plane, resulting in asharp in-focus image when the particle adheres to surface 140 b. Duringspinning, if the centrifugal force F is sufficiently strong to causebond rupture, particle 240 is quickly (typically at a speed of microns/sor faster) pulled away by centrifugal force F from surface 140 b (e.g.,along z-axis of FIG. 1). The escape of the particle from the objective'sfocal plane can be observed by changes in the characteristics of thedetected image that are correlated with changes in focal depths. Forexample, a disappearance or blurriness of a circular patternrepresenting particle 240 (e.g., similar to the disappearance of object316 from image 320 or the blurriness of object 314′ in image 320)indicates that the particle is being pulled away from the focal plane ofobjective 150, e.g., as a result of bond rupture. Once bond rupture isidentified, the bond force can be computed based on the magnitude ofcentrifugal force F, as defined in equation (1) or (2).

In examples where bond rupture is not necessarily observed or desired,detecting particle movement can provide valuable information about thecompliance of a molecular tether that includes bond 243. For example,the displacement of particle 240 subjected to varying amplitude ofcentrifugal force F can be used to compute the elastic modulus of themolecular tether, and possibly to map the force dependency of suchmodulus. Also, stepwise changes in the position of particle 240 mayindicate the transitions between molecular states that are associatedwith conformational changes of a chemical bond. For example, a bead thatremains tethered but moves suddenly away from the cover glass cansignify unfolding of a protein domain.

The transmitted light imaging technique described above can easilyprovide a spatial resolution along the optical axis of about 100 nm forobservation. In certain implementations where a higher resolution isdesirable, more sophisticated imaging and image processing techniquescan also be applied. For example, by using advanced techniques such asusing transmitted or reflected light interference patterns of individualbeads, the position of a bead relative to surface 140 b can bedetermined with sub-nanometer accuracy.

Referring to FIGS. 4A-4C, the interference patterns of a single particleimaged by a reflection interference contrast microscope (RICM) at threedifferent heights are shown, respectively. In these figures, thealternation of dark and bright fringes as well as the intensity and thesize of each fringe can be decoded to reconstruct the height profile ofthe particle with a sub-nanometer axial resolution. (The white bar shownin these figures represents 10 μm). An example of particle trackingusing the RICM technique is described by Heinrich et al., in FastThree-Dimensional Tracking of Laser-Confined Carrier Particles,published in Langmuir, 24(4):1194-1203, 2008, the contents of which areincorporated herein by reference.

In addition to the aforementioned imaging techniques, fluorescencetechniques can also be implemented alone or together withtransmitted/reflected light techniques to enhance resolution and toenable visualization of subtle molecular transitions during experiment.

3 Applications

Using the centrifugal force (and possibly other forces) of a spinningsample, spinning force system 100 is capable of performingsingle-molecule experiments for application in a wide range of areas,including receptor-ligand interactions, DNA mechanics, the kinetics ofmotor proteins, and the dynamics of intramolecular transitions such asprotein folding and unfolding. In addition, spinning force system 100can also be used for high-throughput molecular screening in whichthousands of single-molecule experiments of the same or different typescan be performed in parallel and/or in series. This can be particularlyuseful for drug discovery and screening. The following section providesseveral scenarios in which spinning force system 100 is useful.

3.1 Example I

One application of spinning force system 100 relates to studying themolecular interactions between two or more interacting molecules ormolecular complexes, including, for example, measuring the associationrate K_(on) and dissociation rate K_(off) of the interaction,identifying metastable states, and determining the transition ratesbetween such states. Examples of interacting molecules suitable assubject of study include receptor-ligand pairs, such asbiotin-streptavidin, antibody-antigen, enzyme-substrate, andDNA-polymerase.

Referring to FIGS. 5A-5D, one procedure of preparing a sample containingtwo interacting molecules A (e.g., biotin) and B (e.g., streptavidin)for measurement is illustrated.

For example, in FIG. 5A, a cover glass 510 is coated with a dispersedlayer of molecule A 512 using proper functionalization techniques suchas physisorption or covalent linkage.

In FIG. 5B, a solution of glass beads 520 is then added onto cover glass510. Each bead is pre-coated with one or more molecules B 522, againusing proper functionalization techniques.

In FIG. 5C, cover glass 510 is incubated with glass beads 520 to enableformation of interaction 530 between molecules A and B. Consequently,some beads 520 become attached to the surface of cover glass 510 throughnewly formed interactions 530. Unattached beads can be removed from thesurface by rinsing with an inactive solution, or alternatively, removedby a centrifugal force applied during measurement. In some examples, theamount of interactions 530 formed during the incubation can becontrolled, for example, by the temperature and time duration of theincubation, the concentration of pre-coated bead solution, and thedensity of molecule A 512 seeded on cover glass 510.

In FIG. 5D, once the sample is ready for measurement, it is loaded ontospinning force system 100. In this example, cover glass 510 isconfigured to be parallel to rotational axis 102 of system 100. Therotation of the sample therefore results in a centrifugal force F onbead 520, pulling it away in a direction perpendicular to the surface ofcover glass 510. The movement of bead 520 can be monitored using one ormore of the imaging techniques described above. When the magnitude ofcentrifugal force F is sufficiently high to rupture interaction 530,bead 520 is released from cover glass 510.

In this example, hundreds or thousands of molecular interactions 530 canbe formed and observed in one experiment, allowing statistical analysisof such observations to be performed in a highly efficient manner.

3.2 Example II

Another application of spinning force system 100 relates to studying theintramolecular dynamics of a single molecule or a molecular tether(e.g., a protein or a DNA tether), including, for example, measuring thefolding, unfolding, stretching, and relaxation of a molecular strand.

FIGS. 6A-6D illustrate use of system 100 in a procedure for preparing asample containing a molecular tether to be measured.

In FIG. 5A, a cover glass 610 is coated with a dispersed layer ofmolecule A 612 using proper functionalization techniques such asphysisorption.

In FIG. 6B, a solution of glass beads 620 and a solution of moleculartethers 630 (e.g., DNA tethers as shown in FIGS. 6B-6D for illustrativepurposes) are added onto cover glass 610. Each bead 620 is pre-coatedwith one or more molecule A 612, and each molecular tether 630 isfunctionalized with one or more molecule B 632 capable of forminginteractions with molecule A 612. Examples of commonly usedfunctionalization techniques and procedures include silanization forglass substrate, formation of a peptide bond between a carboxylatedsurface and a free amine on a protein, and formation of disulfide bondsor S—C bonds for cysteine residues.

In FIG. 6C, cover glass 610 is incubated with glass beads 620 andmolecular tether 630 to enable formation of interaction 640 betweenmolecules A and B. Consequently, some beads 620 become attached to thesurface of cover glass 610 through a newly formed interaction 640between bead 620 and molecular tether 630 and another newly formedinteraction 640′ between molecular tether 630 and cover glass 610.Unattached beads can be removed from the surface by rinsing with aninactive solution, or alternatively, removed by a centrifugal forceduring measurement. In some examples, the amount of interactions 640formed during incubation can be controlled, for example, by thetemperature and time duration of incubation, the concentration ofpre-coated bead solution and molecular tether solution, and the densityof molecule A 612 seeded on cover glass 610.

In FIG. 6D, once the sample is ready for measurement, it is loaded ontospinning force system 100. For illustrative purposes, cover glass 610 isconfigured to be parallel to rotational axis 102 of system 100. Therotation of the sample therefore results in a centrifugal force F onbead 620, pulling it away in a direction perpendicular to the surface ofcover glass 610. The movement of bead 620 indicates the force-inducedconformational changes of molecular tether 630 and can be used tomeasure variables characterizing the folding, unfolding, stretching andrelaxation of the molecular tether in response to an external force.When the magnitude of F is varied (without exceeding the bond ruptureforce to break interaction 634), the force dependent dynamics of DNAscan also be quantified.

Again, in this example, hundreds or thousands of molecular tethers 630can be studied in a single experiment, allowing statistical analysis ofthe results to be performed efficiently.

3.3 Example III

Another application of spinning force system 100 relates to studying thecharacteristics of molecules or molecular interactions in controlledchemical environments, including, for example, quantifying moleculardynamics at various temperatures, pH conditions, and/or saltconcentrations, as well as in the presence of various kinds ofsurfactants and/or enzymes.

FIGS. 7A-7D illustrate use of system 100 in a procedure for preparing asample containing a molecular tether 730 that can be modified by arestriction enzyme 760.

For example, in FIG. 7A, molecular tether 730 (e.g., a DNA strand) isattached to a cover glass 710 and a particle 720 using preparationtechniques described above with reference to FIGS. 6A-6C.

In FIG. 7B, a solution of restriction enzymes 760 is added onto coverglass 710. Examples of restriction enzyme 760 include an enzyme thatcuts double-stranded or single-stranded DNA at specific recognitionnucleotide sequences (known as restriction sites). Restriction enzymescan be used for manipulating DNA in various applications such as DNAdigestion and gene insertion.

In FIG. 7C, molecular tether 730 is incubated in the solution ofrestriction enzymes 760 to allow one or more enzyme molecules to bind tothe restriction sites of tether 730.

In FIG. 7D, upon binding with tether 730, restriction enzyme 760 makesincisions at selected locations in tether 730, cutting the tether intotwo or more disconnected pieces. As a result, particle 720 is releasedfrom the surface of cover glass 710. Particle release can be observedusing the imaging techniques described above.

Some or all of the procedures shown in FIGS. 7A-7D may be performedduring spinning. For example, the binding/interaction between tether 730and restriction enzyme 760 may be monitored under the influence of acontrolled centrifugal force applied to particle 720 as shown in FIG.6D.

3.4 Example IV

Another application of spinning force system 100 relates to studyingdifferent types of molecular interactions and/or molecular dynamics inparallel. This application allows a direct comparison of molecularevents of similar or different nature, such as the mobility of twodifferent motor proteins, the compliance of various pieces of DNAstrands, and the affinity of various receptor-ligand pairs. Todistinguish the observations of motion characteristics resulted fromdistinct molecular events, each type of event can be uniquely labeled,for example, using fluorescent tracers or other kinds of markers. Forexample, in cases where two types of cytoskeletal proteins (e.g.,microtubules and actin filaments) are studied at once, each type can belabeled by fluorescent beads of distinct emission wavelengths. By usingselected optical filters to alternate observation of photons ofdifferent colors, the motion characteristics of each type ofcytoskeletal proteins can be distinguished based on the resultingfluorescent images.

3.5 Example V

Although the previous examples are provided primarily in the context ofmeasuring molecular dynamics or interactions, spinning force system 100is also useful in measuring characteristics of interactions that occuron a cellular and/or tissue level. One example is to study the adhesionstrength between adherent cells (e.g., endothelial or epithelial cells)and the underlying substrate or matrix to which the cells adhere. Amonolayer of adherent cells can be seeded on a receptor-modified (e.g.,fibronectin-coated) cover glass to form adhesion complexes (e.g.,clusters of adhesion molecules such as integrins, syndecans, andcadherins). The cover glass is then mounted to rotary arm 120 and cellsare monitored during rotation. The magnitude of centrifugal force thatdetaches cells from the cover glass indicates the maximum adhesionstrength of the adhesion complexes.

3.6 Example VI

Another application of spinning force system 100 relates to using system100 in conjunction with other detection or measurement techniques (e.g.,various kinds of single-molecule force probes or fluidic systems) toobtain force-related characteristics of the sample. For example, system100 can be combined with a magnetic system to apply both magnetic andcentrifugal forces to manipulate a test subject. A biotin labeledmagnetic bead can be first pulled by a magnetic force toward a cellmembrane (or a streptavidin-treated surface) to form abiotin-streptavidin attachment. The strength of this attachment can thenbe tested by applying a centrifugal force that pulls the bead away fromthe membrane.

System 100 can also be combined in use with devices that apply varioustypes of chemical stimuli (e.g., chemoattractants and enzymes) andmechanical stimuli (e.g., compression, extension, and shearing) to atest subject, simulating the native environment of the subject duringobservation. For example, when system 100 is used for measuring themechanical properties (e.g., elasticity or viscoelasticity) ofendothelial cells, during spinning, a laminar or cyclic shear stress mayalso be provided to mimic the conditions of the endothelial cells invivo. Additionally, cellular response to chemical/mechanical stimuli mayalso be observed.

3.7 Other Examples

In addition to detecting characteristics of biomolecular or chemicalinteractions, system 100 is also suitable for use with a wide range ofgeneral testing tasks. Examples include 1) measuring the mechanicalproperties of polymer networks or aggregates, where the overall behaviorof the network may be of interest; 2) testing the strength and othercharacteristics of physical bonds formed between a subject and asurface; and 3) observing the behavioral changes of test subjectseffected by chemical agents (e.g., an enzyme slicing a single DNAbetween two surfaces or urea denaturing a protein).

4 Alternative Embodiments

Many variations of spinning force system 100 are possible.

Referring to FIG. 8, an alternative embodiment of spinning force system800 includes a coaxial detection assembly 820 mechanically coupled to arotary stage 810. Detection assembly 820 includes a LED lamp 830, asample holder 840, an objective lens 850, and a CCD camera 860, eachbeing coaxially aligned with optical axis 870. LED lamp 830 isintegrated into sample holder 840, which can be translated (e.g., alongoptical axis 870) through a focus knob for coarse adjustment. Fineadjustment of the position of sample holder 840 can be performed using apiezoelectric stage coupled to the holder (not shown). When rotary stage810 operates in circular motion at angular velocity ω, the entiredetection assembly 820 rotates at ω.

In some implementations, detection assembly 820 may be miniaturized tofit inside a standard centrifuge, eliminating the need for rotary stage810 and reducing system cost.

Referring to FIG. 9, another alternative embodiment of spinning forcesystem 900 is shown. Unlike detector 160 in system 100, detector 960 inthis example is not mounted on rotary arm 920. Rather, detector 960remains immobile during operation of system 900. Light from objective950 are directed by a rotating mirror 970 to detector 960 through lightpaths 952 and 954 subsequently. Rotating mirror 970 is not necessarilymounted on rotary arm 920. For example, mirror 970 can be coupled to aseparate rotor (not shown) or directly coupled to rotary stage 910 inorder to reflect light from the rotating objective 950 to the stationarydetector 960.

In the above examples of FIGS. 1, 8, and 9, the samples are detectedusing trans-illumination imaging techniques. More specifically, in FIG.1, light 132 emitted from LED 130 transmits (penetrates) through sample140 prior to being received by objective 150. In other examples,epi-illumination imaging techniques can also be conveniently used.

Referring to FIG. 10, a further alternative embodiment of spinning forcesystem 1000 with epi-illumination is shown. Here, a Dichroic mirror 1070is positioned between detector 1060 and objective 1050, serving as abeam splitter that directs light beams of different characteristics(e.g., different wavelengths) onto different paths. For example, lightbeam 1032 from LED 1030 is first deflected by Dichroic mirror 1070towards objective 1050 (along light path 1032′) to illuminate a selectedregion of sample 1040. A light beam 1034 is reflected from sample 1040as a result of the illumination, travelling in a direction opposite tolight beam 1032′. After passing through objective 1050 and DicroicMirror 1070, light beam 1034 is received by detector 1060 to produce animage of the illuminated region of sample 1040. In this example,objective 1050 serves as both a light condenser and an imaging lens.

In some embodiments, a vertical swinging or radially movable arm may beintegrated to system 100 to enhance the flexibility of force control.System 100 may also be multiplexed to have many arms, each configured tocarry a unique experiment. Each arm may also have a different andpossibly adjustable length to facilitate force control.

It is to be understood that the foregoing description is intended toillustrate and not to limit the scope of the invention, which is definedby the scope of the appended claims. Other embodiments are within thescope of the following claims.

What is claimed is:
 1. A method comprising: attaching a particle to asurface of a substrate through a molecular interaction associated with afirst molecule and a second molecule; rotating the substrate to apply aforce to the particle in a direction that is not in parallel to thesurface of the substrate; detecting an image of the particle with adetector during rotation of the substrate, the image containinginformation representing a characteristic of the molecular interaction;and determining the characteristic of the molecular interaction based onthe detected image.
 2. The method of claim 1, further comprising:detecting a relative motion of the particle to the surface of thesubstrate.
 3. The method of claim 2, wherein the relative motionincludes detachment of the particle from the surface of the substrate.4. The method of claim 2, wherein the relative motion includes a motionindicative of a rupture or a formation of a chemical bond associatedwith the molecular interaction.
 5. The method of claim 2, wherein therelative motion includes a motion indicative of a conformational changeof at least one of the first and the second molecules.
 6. The method ofclaim 5, wherein the conformational change includes any one or more ofunfolding, refolding, stretching and relaxing of at least one of thefirst and second molecules.
 7. The method of claim 1, whereindetermining the characteristic of the molecular interaction includesmeasuring a strength characteristic of the molecular interaction.
 8. Themethod of claim 1, wherein determining the characteristic of themolecular interaction includes measuring a kinetic characteristic of themolecular interaction.
 9. The method of claim 8, wherein the kineticcharacteristic of the molecular interaction includes an association rateK_(on) or a dissociation rate K_(off) of the molecular interaction. 10.The method of claim 1, further comprising forming a first chemicallinkage between the particle and the first molecule.
 11. The method ofclaim 1, further comprising forming a second chemical linkage betweenthe second molecule and the surface of the substrate.
 12. The method ofclaim 1, further comprising controlling a rotation of the substrate. 13.The method of claim 12, wherein controlling a rotation of the substratefurther includes controlling an angular velocity of the rotation of thesubstrate.
 14. The method of claim 1, wherein the first molecule is areceptor and the second molecule is a ligand of the receptor.
 15. Themethod of claim 1, wherein the first molecule includes a first molecularcomplex, and the second molecule includes a second molecular complexthat interacts with the first molecular complex.
 16. The method of claim15, wherein the first molecular complex includes one or more pairs ofreceptor and ligand.
 17. The method of claim 1, wherein the particleincludes a bead.
 18. The method of claim 17, wherein the bead has adiameter in the range between 1 nm and 1 mm, and a density in the rangebetween 0.01 g/cm³ and 20 g/cm³.
 19. A method comprising: attaching aparticle to a surface of a substrate through a molecular interactionassociated with a first molecule and a second molecule; rotating thesubstrate to apply a force to the particle in a direction that is not inparallel to the surface of the substrate; rotating at least a portion ofa detection assembly with the substrate; detecting an image of theparticle with the detection assembly, the image containing informationrepresenting a characteristic of the molecular interaction; anddetermining the characteristic of the molecular interaction based on thedetected image.
 20. The method of claim 19, wherein the detectionassembly comprises a detector, and wherein the substrate is rotatedrelative to the detector.
 21. A method comprising: attaching a particleto a surface of a substrate through a molecular interaction associatedwith a first molecule and a second molecule; rotating the substrate toapply a force to the particle in a direction that is not in parallel tothe surface of the substrate; detecting an image of the particle with adetector during an event associated with the molecular interaction, theevent being caused by rotation of the substrate and the image containinginformation representing a characteristic of the molecular interaction;and determining the characteristic of the molecular interaction based onthe detected image.
 22. The method of claim 21, wherein the eventassociated with the molecular interaction comprises detachment of theparticle from the substrate.
 23. The method of any one of claim 1, 19 or21, wherein the molecular interaction is a molecular interaction betweenthe first molecule and the second molecule.
 24. The method of any one ofclaim 1, 19 or 21, wherein the molecular interaction is a molecularinteraction within at least one of the first and second molecules. 25.The method of any one of claim 1, 19 or 21, wherein the characteristicof the molecular interaction is a rupture or a formation of a bond in atleast one of the first and second molecules.
 26. The method of claim 25,wherein the characteristic of the molecular interaction is a rupture ora formation of a bond in the presence of an enzyme.
 27. The method ofany one of claim 1, 19 or 21, wherein the characteristic of themolecular interaction is a conformational change of at least one of thefirst and second molecules.
 28. The method of claim 27, wherein theconformational change includes any one or more of unfolding, refolding,stretching and relaxing of at least one of the first and secondmolecules.
 29. The method of any one of claim 1, 19 or 21, wherein morethan one image is detected.
 30. The method of any one of claim 1, 19 or21, wherein at least one of the first and second molecules is DNA. 31.The method of any one of claim 1, 19 or 21, wherein at least one of thefirst and second molecules is a protein.
 32. A method comprising:attaching a particle to a surface of a substrate, wherein the particleis attached to a first molecule; rotating the substrate to apply a forceto the particle in a direction that is not in parallel to the surface ofthe substrate; detecting an image of the particle with a detector duringrotation of the substrate, the image containing information representinga characteristic of the first molecule; and determining thecharacteristic of the first molecule based on the detected image. 33.The method of claim 32, wherein the characteristic of the first moleculeis a molecular interaction within the first molecule.
 34. The method ofclaim 32, wherein the characteristic of the first molecule is a ruptureor a formation of a bond in the first molecule.
 35. The method of claim32, wherein the characteristic of the first molecule is a rupture or aformation of a bond in the presence of an enzyme.
 36. The method ofclaim 32, wherein the characteristic of the first molecule is aconformational change in the first molecule.
 37. The method of claim 36,wherein the conformational change includes any one or more of unfolding,refolding, stretching and relaxing of the first molecule.
 38. The methodof claim 32, wherein more than one image is detected.
 39. The method ofclaim 32, wherein the first molecule is DNA.
 40. The method of claim 32,wherein the first molecule is a protein.